Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Conventional macromolecule delivery and release technologies, which in the past have concentrated on improvements in mechanical devices such as implants or pumps to achieve more targeted and sustained releases of drugs, is now advancing on a microscopic and even molecular level. Recombinant technology has produced a variety of new potential therapeutics in the form of nucleic acids, proteins and peptides and these successes have driven the search for newer and more flexible macromolecule delivery and targeting methods and systems.
Microencapsulation of different molecules within biodegradable polymers and lipid complexes has achieved successes in improving the targeting and delivery of a variety of molecules including nucleic acids and various chemotherapeutic agents. For example, lipid complexes are currently used as delivery vehicles for a number of molecules where sustained release or target release to specific biological sites is desired. In the case of nucleic acids, charged nucleic acid-lipid complexes are utilized to enhance transfection efficiencies in somatic gene transfer by facilitating the attachment of nucleic acids to the targeted cells.
Success in somatic gene therapy depends on the efficient transfer and expression of extracellular DNA to the nucleus of eucaryotic cells, with the aim of replacing a defective or adding a missing gene (1). Viral-based carriers of DNA are presently the most common method of gene delivery, but there has been a tremendous activity in developing synthetic nonviral vectors. In particular, cationic liposomes (CLs), in which the overall positive charge of the cationic liposome-DNA (CL-DNA) complex enhances transfection by attaching to anionic animal cells, have shown gene expression in vivo in targeted organs, and human clinical protocols are ongoing (2-4). Cationic liposome transfer vectors exhibit low toxicity, nonimmunogenicity, and ease of production, but their mechanism of action remains largely unknown with transfection efficiencies varying by up to a factor of 100 in different cell lines (2-6).
This unpredictability, which is ubiquitous in gene therapy (7) and in particular in synthetic systems, may be attributed to a lack of knowledge regarding the interactions between DNA and CLs and the resulting structures of CL-DNA complexes. DNA membrane interactions might also provide clues for the relevant molecular forces in the packing of DNA in chromosomes and viral capsids. Studies show regular DNA condensed morphologies induced by multivalent cations (8) and liquid-crystalline (LC) phases at high concentrations of DNA both in-vitro (9) and in-vivo in bacteria (10). More broadly, the nature of structures and interactions between membranes and polymers, either adsorbed (11) or tethered to the membranes ( 12), is currently an active area of research.
Felgner et al. (3) originally proposed a xe2x80x9cbead-on-stringxe2x80x9d structure of the CL-DNA complexes picturing the DNA strand decorated with distinctly attached liposomes. Electron microscopy (EM) studies have reported on a variety of structures including string-like structures and indications of fusion of liposomes in metal-shadowing EM (13), oligolamellar structures in cryo-TEM (14), and tube-like images possibly depicting lipid bilayer-covered DNA observed in freeze-fracture EM (15).
A variety of modifications of the lipid membranes have been attempted with limited success, including polymerizing or crosslinking the molecules in the bilayer to enhance stability and reduce permeation rates, and incorporating polymers into the bilayer to reduce clearance by macrophages in the bloodstream. While these modifications have proved beneficial, without means to overcome the inherent unpredictability of these complexes by controlling crucial factors such as lipid membrane thickness and the intermolecular spacing of the encapsulated molecules, the use of these molecules is severely limited. The present invention is directed to overcoming this limitation.
The invention provides novel compositions involving macromolecule-lipid complexes and methods for making them. These compositions and methods of the invention are significant improvements in the field of macromolecule-lipid complex synthesis, macromolecule targeting and delivery to various biological systems.
The present invention provides methods for making macromolecule-lipid complexes and methods for controlling components of the macromolecule-lipid complexes such as the membrane thickness and intermolecular spacing of the complex constituents.
In one embodiment for making macromolecule-lipid complexes, the method comprises mixing a lipid combination (e.g., a neutral lipid and a charged lipid) in a sufficient amount with a macromolecule so as to form a complex with specific geometric and charge qualities. By varying the relative amounts of (1) the charged and neutral lipids, (2) the weight amount and/or the macromolecule and (3) the assembly solution, conditions distinct complexes can be generated having desired isoelectric point or charged states.
By utilizing this process for controlling both the exterior lipid structure and interior macromolecular ordering, an extremely versatile molecular targeting and delivery system can be developed for a variety of applications. The invention has applications in the numerous methods which utilize lipids and various macromolecules such as gene therapy, nucleic acid based vaccine development and peptide and protein delivery.